An ability to sense and respond to diverse stress stimuli from the environment is a basic cellular function conserved throughout evolution. In unicellular organisms, such responses are critical for survival. In higher organisms including humans, cellular stress responses can be seen in the inflammation resulting from asthma, arthritis, or bacterial infection, as well as in the response of cancer cells to cytotoxic therapies. In order to sense stress stimuli and regulate cellular physiology, eukaryotic organisms from yeast to humans utilize a signaling module called a MAP kinase (MAPK) cascade. MAPKs dedicated for stress signaling are also known as SAPKs (Stress-Activated Protein Kinases) and play key roles in cellular responses to changes in the environmental conditions as well as responses to bacterial endotoxins, inflammatory cytokines, and chemotherapeutic drugs. The long-term objective of the research described in this proposal is to understand at a molecular level how diverse stress stimuli are sensed and transmitted to SAPK, and how activated SAPK then modulates cellular processes for stress adaptation. These studies will use the genetically tractable model system provided by the fission yeast Schizosaccharomyces pombe, whose SAPK pathway has been demonstrated to be structurally and functionally similar to those in humans. Signaling within MAPK cascades is achieved by sequential activation of a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and finally, a MAPK. In the S. pombe SAPK cascade, signaling to the Spcl SAPK is initiated by two MAPKKKs, Wis4 and Win1. We have found that Wis4 and Win1 form a complex with multiple proteins and serve as a hub for stress sensing and response via the Spc1 pathway. The three specific aims focus on this MAPKKK complex: (I) to determine how the complex is organized and discover novel proteins that interact with Wis4 and Win1; (II) to uncover how oxidative stress signaling to the SAPK cascade is mediated by a glycolytic enzyme, GAPDH, which has unexpectedly been found as a component of the MAPKKK complex; and (iii) to determine how another MAPKKK-interacting protein, Wsh3, regulates a newly discovered cellular function of the SAPK pathway, the maintenance of cell polarity under high similarity stress. It is anticipated that the proposed SAPK research in S. pombe will serve as a valuable paradigm for human SAPK, facilitating understanding of the roles of SAPKs in clinical contexts.